A "scan and stop" assay identifies CD40 and CD70 as selective regulators of T-cell arrest on APCs.

T-cell activation requires firm arrest on APCs, a process essential for effective clonal expansion and differentiation. Although the role of co-inhibitory signals and integrin-mediated adhesion in modulating T-cell arrest is established, the contribution of co-stimulatory molecules to this process remains poorly understood. Here, we developed a quantitative "scan and stop" assay using engineered CHO cells as minimalistic APCs to systematically assess the influence of co-stimulatory proteins on T-cell arrest. These APCs express only selected peptide-MHC complexes and co-stimulatory ligands, allowing controlled investigation of their roles in both naïve and experienced CD4+ and CD8+ T cells. We found that CD40 selectively promotes the arrest of pre-activated CD4+ T cells, whereas CD70 enhances the arrest of CD8+ T cells, correlating with expression patterns of their respective receptors, CD40L and CD27. High-resolution imaging further revealed mechanical deformation of APCs during synapse formation, suggesting force generation by T cells. Altogether, our results identify CD40 and CD70 as subtype-specific regulators of T-cell arrest and reveal a novel dimension in co-stimulatory control of immune synapse formation.